The invention relates to reagents and methods for characterizing receptor-ligand interactions, identifying modulators of such interactions, and characterizing the receptor expression profile of cells and cell populations. The invention finds application in the biomedical sciences.
Communication between cells is a fundamental process involved in growth, differentiation, metabolism, and generation of biological responses (e.g., immune responses) in multicellular organisms. Often, cell-to-cell signaling is mediated by extracellular receptors or cell adhesion molecules. These membrane-associated molecules interact with other proteins, such as soluble factors such as peptide hormones, extracellular matrices, and cell surface molecules displayed by other cells. In some cases, signaling involves cell-to-cell contact (e.g., contact between two surface proteins on cells). An example of such an interaction is the binding of the T cell receptor on the surface of a T lymphocyte and the MHC/antigen complex on the surface of an antigen-presenting cell. A different type of cell-to-cell signaling is mediated by soluble polypeptides that, by interacting with a specific receptor on a target cell, lead to changes in target cell activity. An illustrative and important example cell-to-cell signaling mediated by soluble polypeptides is the activity of chemokines in mammalian systems.
Chemokines are a class of cytokines that play an important role in inflammatory responses, leukocyte trafficking, angiogenesis, and other biological processes related to the migration and activation of cells. As mediators of chemotaxis and inflammation, chemokines play roles in pathological conditions. For example, the concentration of chemokine MCP-1 is higher in the synovial fluid of patients suffering from rheumatoid arthritis than that of patients suffering from other arthritic diseases.
Known chemokines are typically assigned to one of four subfamilies based on the arrangement of cysteine motifs. In the so-called alpha-chemokines, for example, the first two of four cysteines (starting from the amino terminus) are separated by an intervening amino acid (i.e., having the motif C-X-C). The beta-chemokines are characterized by the absence of an intervening amino acid between first two cysteines (i.e., comprising the motif Cxe2x80x94C). The smaller gamma-chemokine family is characterized by a single C residue (gamma). The delta-chemokine family is characterized by a pair of cysteines separated by three residues (i.e., having the motif CX3C). The sole CX3C chemokine (fractalkine) is a type 1 membrane protein containing a chemokine domain tethered on a long mucin-like stalk. Fractalkine, also named neurotactin, has contains a chemokine domain at the amino terminus tethered on a long mucin-like stalk. For a recent review on chemokines, see Ward et al., 1998, Immunity 9:1-11 and Baggiolini et al., 1998, Nature 392:565-568, and the references cited therein.
Some chemokine activities (e.g., promigratory effects) are mediated by binding to an array of cell surface receptors on the surface of target leukocytes. These receptors are of the seven transmembrane spanning, G protein coupled receptor class (alternately referred to as 7TM or GPCR). Several seven-transmembrane-domain G protein-coupled receptors for Cxe2x80x94C chemokines have been cloned: a Cxe2x80x94C chemokine receptor-1 which recognizes MIP-1xcex1, RANTES, MCP-2, MCP-3, and MIP-5 (Neote et al., 1993, Cell, 72:415-415); CCR2 which is a receptor for MCP1, 2, 3 and 4 or 5; CCR3 which is a receptor for RANTES, MCP-2, 3, 4, MIP-5 and eotaxin; CCR5 which is a receptor for MIP-1xcex1, MIP-1xcex2 and RANTES; CCR4 which is a receptor for CMDC or TARC; CCR6 which is a receptor for LARC; and CCR7 which is a receptor for SLC and MIP-3xcex2 (reviewed in Sallusto et al., 1998, Immunol. Today 19:568 and Ward et al., 1998, Immunity 9:1-11).
Due to the importance of interactions between receptors (such as chemokine receptors) and their ligands in biological function, a need exists for rapid and effective methods for the characterization of such interactions.
In one aspect the invention provides an assay device having a plurality of different immobilized tethered ligand fusion proteins organized in an array, where the ligand-stalk fusion proteins include a ligand domain and a stalk domain (xe2x80x9cassay devicexe2x80x9d). In one embodiment, the ligand-stalk fusion proteins have a ligand domain, an intermediate stalk domain, and an immobilization domain (xe2x80x9cassay devicexe2x80x9d). In an embodiment, the ligand domain and the stalk domain are not associated in a naturally occurring protein. In various embodiments, the immobilized tethered ligand fusion proteins include a mucin-derived stalk sequence, a fractalkine mucin repeat region sequence, and/or a ligand domain encoding a chemokine.
In another aspect, the invention provides a tethered ligand fusion protein, where the ligand domain is other than a chemokine sequence.
In another aspect, the invention provides a method for identifying an interaction between a receptor and a ligand by contacting a cell expressing a receptor or a ligand-binding portion thereof with an immobilized tethered ligand fusion protein and, detecting binding of the cell and the tethered ligand fusion protein, where binding of the cell to the fusion protein is correlated with an interaction between the receptor and the ligand corresponding to the ligand domain of the fusion protein. In an embodiment, the tethered ligand is immobilized on an assay device, supra. In various embodiments, the cell expresses a recombinant receptor (e.g., an orphan receptor). In an embodiment, the cell expresses a chemokine receptor. In one embodiment, an interaction between a receptor and more than one ligand is detected.
In another aspect, the invention provides a method for identifying a modulator of an interaction between a receptor and a ligand by contacting cells expressing the receptor or a ligand-binding portion thereof with an immobilized tethered ligand fusion protein in the absence of a test compound, and measuring binding of the cells to an immobilized tethered ligand fusion protein, contacting cells expressing the receptor or a ligand-binding portion thereof with an immobilized tethered ligand fusion protein in the presence of a test compound, and measuring binding of the cells to the immobilized tethered ligand fusion protein; and comparing the levels of binding, where decreased binding of cells in the presence of the test compound indicates that the test compound is an inhibitor of the interaction between the receptor and the ligand corresponding the ligand domain of said fusion protein, and where increased binding of cells in the presence of the test compound indicates that the test compound is an enhancer of the interaction between the receptor and the receptor and the ligand corresponding the ligand domain of the fusion protein. In an embodiment, the tethered ligand is immobilized on an assay device, supra. In one embodiment, the receptor is a chemokine receptor.
In another aspect, the invention provides a method for detecting a profile of receptor expression in cells in a population of cells by contacting the population of cells with an immobilized tethered ligand fusion protein and detecting binding of cells of the population to the tethered ligand fusion protein, where binding of a cell in the cell population to a tethered ligand fusion protein is correlated with expression of a receptor that binds the ligand corresponding to the ligand domain of the fusion protein. In an embodiment, the tethered ligand is immobilized on an assay device, supra. In an embodiment, the contacting step involves contacting the population with a plurality of different fusion proteins and the detecting step involves detecting binding of cells to zero, one or more than one fusion proteins. In one embodiment, the population is heterogeneous, e.g., the population is obtained from synovial fluid, cerebral-spinal fluid, bronchial alveolar lavage (BAL) fluid, or blood. In an embodiment, the method includes quantitating the level of binding to each tethered ligand fusion protein in the array, or characterizing the cells bound at each sector of the array (e.g., by immunostaining).
In another aspect, the invention provides a method for diagnosis. The method includes the steps of obtaining a population of cells from a patient suspected of suffering from a disease, determining a receptor profile for the population, and, comparing said receptor profile with a profile characteristic of the disease state. In one embodiment the receptor profile is determined by contacting the population of cells with an immobilized tethered ligand fusion protein and identifying a tethered ligand fusion protein bound by cells of said population thereby identifying a receptor profile. In an embodiment, the tethered ligand is immobilized on an assay device, supra. In various embodiments, determining the method also includes quantitating the binding of cells at each sector of the array or characterizing the cells bound at each sector of the array, e.g., by immunostaining. In various embodiments, the disease is an inflammatory or allergic disease, or an autoimmune disease. In various embodiments, the population is obtained from synovial fluid, cerebral-spinal fluid, bronchial alveolar lavage (BAL) fluid, or blood.
In another aspect, the invention provides a method for detecting an effect of a drug or treatment on a patient by determining the receptor profile of a population of cells from the patient for the first time, administering the drug or treatment to the patient, determining the receptor profile of a population of cells from the patient for a second time, and comparing the receptor profiles obtained to determine the effect of the drug or treatment receptor-expressing cells in the patient. In an embodiment, the determining is carried out by contacting said population of cells with an immobilized tethered ligand fusion protein and identifying a subset of arrayed tethered ligand fusion proteins bound by cells of said population thereby identifying a receptor profile. In an embodiment, the tethered ligand is immobilized on an assay device, supra. In various embodiments, determining the method also includes quantitating the binding of cells at each sector of the array or characterizing the cells bound at each sector of the array, e.g., by immunostaining. In various embodiments, the population is obtained from synovial fluid, cerebral-spinal fluid, bronchial alveolar lavage (BAL) fluid, or blood.
In another aspect, the invention provides a method for identifying a modulator of an interaction between a receptor and a ligand by contacting cells expressing the receptor or a ligand-binding portion thereof with an immobilized tethered ligand fusion protein in the presence of a test compound and determining the level of binding to the fusion protein, contacting cells expressing the receptor or a ligand-binding portion thereof with the immobilized tethered ligand fusion protein in the absence of a test compound; and determining the level of binding to the fusion protein, and comparing the level of binding, where the ligand-stalk fusion proteins include a ligand domain, an intermediate stalk domain, and, an immobilization domain and where decreased binding of cells in the presence of the test compound indicates that the test compound is an inhibitor of the interaction between the receptor and the ligand corresponding the ligand domain of fusion protein, and where increased binding of cells in the presence of the test compound indicates that the test compound is an enhancer of the interaction between the receptor and the ligand corresponding the ligand domain of the fusion protein. In one embodiment, the ligand domain and the stalk domain are not associated in a naturally occurring protein. In an embodiment, the stalk domain is carboxy-terminal to the ligand domain, and the immobilization domain is carboxy-terminal to the stalk domain.
In another aspect, the invention provides the use of tethered ligand fusion proteins in any of the methods described herein.